Fig. 5: The Swd2 WD40 domain is critical for COMPASS function.
From: The Set1 N-terminal domain and Swd2 interact with RNA polymerase II CTD to recruit COMPASS
a Swd2–HA or Swd2–F250A–HA were expressed in an swd2Δ deletion strain. Protein levels were analyzed by immunoblotting using the indicated antibodies. Actin and Histone H3 were used as loading controls. Note that samples were run on the same blot, but intervening lanes were removed (dashed line, full blots appear in Source data). b Plasmid-encoded Swd2–HA and Swd2–F250A–HA were expressed in FLAG-Set1 containing cells that also express the endogenous untagged SWD2 gene in the chromosome. Proteins were immunoprecipitated with anti-FLAG beads (IP:FLAG) and then immunoblotted for Set1 and Swd2-HA. c The Y2H interaction between the Gal4BD–Set1 and Gal4AD–CTD fusions was tested in strain PJ69-4A expressing wild-type (WT) or mutant (F250A) Swd2. The empty Gal4 vectors served as negative controls. Activation of the Gal4-activated HIS3 reporter was tested on plates lacking histidine, with the addition of 5 mM 3AT in the last panel for additional stringency. d Analysis of recombinant baculovirus-expressed Set1/COMPASS made with WT Swd2 or the Swd2–F250A mutant. SDS-PAGE/Coomassie blue staining shows total protein, while immunoblotting with anti-FLAG and anti-HA show Set1 and Swd2 levels, respectively. e H2Bub chromatin was subjected to in vitro histone methyltransferase assays16 with Swd2 WT and Swd2-F250A mutant-containing complexes shown in panel (d). H3K4 methylation status was monitored by immunoblotting with indicated antibodies. Histone H3 was used as an internal loading control. Source data are provided as a Source data file.
